Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.6.1.3 (ATPase)
 65,361 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Digoxin is known to be secreted by renal tubular cells, but the mechanisms are still not fully understood. In this study, we examined renal tubular cell handling of digoxin and ouabain using LLC-PK1 cells, a model of proximal renal tubular cells. The cells were used in suspension for binding experiments and in monolayers on permeable filters for transport studies. The specific binding of digoxin to the cells, presumably to the ouabain binding site (i.e., membrane Na+,K(+)-ATPase), were characterized by Kd of 2.6 x 10(-7) M and Bmax (total number of specific binding sites) of 1.6 x 10(6)/cell. Kd and Bmax of ouabain binding were 1.3 x 10(-7) M and 1.9 x 10(6)/cell, respectively. In transport experiments, digoxin showed significantly higher flux than ouabain from the basolateral to the apical side across the cell monolayers. Importantly, this secretory transport was not inhibited by ouabain concentrations sufficient to block membrane Na+,K(+)-ATPase and to displace digoxin from the binding site on the enzyme (i.e., 10(-6) to 10(-4) M ouabain). However, the digoxin secretion was decreased by low temperature or excess digoxin in a concentration-dependent manner. These data suggest that digoxin undergoes unidirectional transport in favor of secretion, which does not involve its binding to the ouabain binding sites on membrane Na+,K(+)-ATPase.
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PMID:Handling of digoxin and ouabain by renal tubular cells (LLC-PK1). 132 Jun 81

Cystinosis is an inherited metabolic disease characterized by accumulation of lysosomal cystine and renal impairment. In an attempt to better understand the link between cystine accumulation and renal functions, we studied the effects of cystine loading on the Na(+)-H+ antiporter and the sodium pump in renal epithelial cells (LLC-PK1) in culture. Incubation of LLC-PK1 with 1 mM cystine dimethyl ester (CDME) for 48 h caused lysosomal cystine loading and reduced by 22 +/- 2% the maximal velocity of sodium-hydrogen antiport with no significant change in the affinity of sodium for the transporter. Rubidium influx decreased to 46 +/- 5% of control. Ouabain binding experiments revealed a 10% reduction in the number of Na(+)-K(+)-ATPase units in the intact cells. Na(+)-K(+)-ATPase activity in the particulate fraction of the cells homogenate declined to 50 +/- 7.5% of controls. No significant change was observed in the activity of ouabain-insensitive phosphatases. The intracellular concentration of sodium increased from 20.6 +/- 3.7 to 64.8 +/- 10 mM, and potassium concentration decreased from 103 +/- 6 to 80 +/- 13 mM. In addition to the observed reduction in the sodium gradient and in agreement with the reduction in the intracellular potassium concentration, the membrane potential changed from -80.8 +/- 7.5 to -69.9 +/- 7.0 mV. The results suggest that intracellular accumulation of cystine is associated with reduction in the number and the activity of membrane transporters. The consequence of the changes in the activity of Na(+)-K(+)-ATPase is a reduction in the electrochemical forces that drive transport in the renal cells tested.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Cystine dimethyl ester reduces the forces driving sodium-dependent transport in LLC-PK1 cells. 132 21

Studies from this laboratory demonstrate that LLC-PK1/Cl4 cells, a cultured renal cell line, respond to incubation in low-K+ medium by coordinately increasing abundance of both alpha- and beta-subunits of Na(+)-K(+)-ATPase but increase only beta- and not alpha-mRNA levels (Lescale-Matys et al. J. Biol. Chem. 265: 17935-17940, 1990) and that alpha-abundance is likely increased as a result of increased efficiency of alpha-mRNA translation (L. Lescale-Matys and A. A. McDonough. J. Cell Biol. 111: 311A, 1990). The aim of this report was to determine if nontransformed kidney cells would respond to low K+ in a similar manner. We incubated primary cultures of rat proximal tubule cells in low K+ (0.25 mM) for up to 24 h to address this aim. Na(+)-K(+)-ATPase activity, measured enzymatically, and abundance of alpha- and beta-subunits, measured by immunoblot, were increased significantly and coordinately by 8 h of low K+, and, by 24 h of low K+, these parameters were increased to 2.17 +/- 0.34 (activity), 2.03 +/- 0.21 (alpha), and 2.39 +/- 0.48 (beta)-fold over control. Pretranslationally, beta-mRNA, measured by Northern blot analysis, increased to 1.76 +/- 0.35 after 3 h of low K+ and to 3.4 +/- 0.75-fold over control after 24 h of low K+. The increase in alpha-mRNA was smaller and delayed compared with the beta-mRNA response, but it was sufficient to account for the observed increase in alpha-protein and Na(+)-K(+)-ATPase activity at steady state: alpha-mRNA increased to 1.27 +/- 0.09 after 6 h and to 1.91 +/- 0.41-fold over control after 24 h in low K+. We conclude that the accumulation of sodium pumps in cultured renal proximal tubule cells, unlike LLC-PK1 cells, can be accounted for by increases in both alpha- and beta-subunit mRNA levels.
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PMID:Low K+ increases Na(+)-K(+)-ATPase alpha- and beta-subunit mRNA and protein abundance in cultured renal proximal tubule cells. 138 Nov 48

Cyclopiazonic acid is a potent inhibitor of calcium uptake and Ca(2+)-ATPase activity in sarcoplasmic and endoplasmic reticulum. In L6 muscle myoblasts, cyclopiazonic acid stimulates the uptake of tetraphenylphosphonium, a lipophilic membrane potential probe, and has antioxidant properties. The purpose of the present study was to investigate the structural requirements necessary for causing the surface charge alterations, and the antioxidant activity in L6 skeletal muscle myoblasts, and for inhibition of calcium transport by rat skeletal muscle sarcoplasmic reticulum vesicles. This was accomplished by comparing the effects of two structurally related tetramic acids, cyclopiazonic acid imine and tenuazonic acid, with cyclopiazonic acid. Cyclopiazonic acid imine inhibited oxalate-assisted 45Ca2+ uptake and ATPase activity in sarcoplasmic reticulum vesicles and stimulated tetraphenylphosphonium accumulation by L6 muscle myoblasts. However, these effects required an approximately fourfold higher concentration than that of cyclopiazonic acid. Tenuazonic acid, up to 1 mM, had no effect on oxalate-assisted 45Ca2+ uptake or Ca(2+)-ATPase activity in sarcoplasmic reticulum vesicles and did not stimulate tetraphenylphosphonium accumulation by L6 muscle myoblasts. Cyclopiazonic acid was only slightly more effective than cyclopiazonic acid imine at preventing the patulin-induced increase in thiobarbituric acid positive substance (used to estimate lipid peroxidation); tenuazonic acid was totally ineffective. Previously, it was shown that cyclopiazonic acid was twice as effective as cyclopiazonic acid imine at preventing increases in thiobarbituric acid positive substance in cultured renal cells, LLC-PK1. Thus, the indole nucleus of cyclopiazonic acid is essential for the membrane-associated biological activity; however, modification of the acetyl group reduces the potency of the activity.
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PMID:Comparison of three tetramic acids and their ability to alter membrane function in cultured skeletal muscle cells and sarcoplasmic reticulum vesicles. 160 18

Basement membrane proteins such as the heparan sulfate proteoglycan (HSPG) are secreted in a polarized fashion from the basolateral membrane of epithelial cells. We have used the microtubule-disrupting drug colchicine to study the role of the microtubule network in directing constitutive secretion to the basolateral membrane of LLC-PK1 renal epithelial cells. Microtubule depolymerization induced by colchicine resulted in fragmentation and redistribution of fluorescently labeled trans-Golgi membranes. Increased immunofluorescent staining of HSPG was associated with these dispersed Golgi cisternae. The biosynthetic processing of HSPG was not significantly altered by the loss of microtubules or by the dispersal of the Golgi elements. The most striking effect of microtubule disruption was the loss of polarity of HSPG secretion. Immunoprecipitation studies showed that HSPG was secreted from both apical and basolateral surfaces of LLC-PK1 cells treated with colchicine, and a similar result was found for the delivery of laminin, another basement membrane protein. In contrast, there was no change in the distribution of an integral basolateral membrane protein, Na(+)-K(+)-ATPase, following colchicine treatment. Our results provide the first demonstration that microtubules are involved in the constitutive trafficking of basolateral secretory proteins. These data also suggest that there may be an inherent difference in the targeting or delivery of membrane and secretory proteins to the basolateral cell surface.
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PMID:Disruption of microtubules alters polarity of basement membrane proteoglycan secretion in epithelial cells. 165 Jan 39

LLC-PK1 cells, an established line from pig kidney, express basolateral and apical Na(+)-H+ exchangers that can be distinguished by their different sensitivities to the amiloride analogue, N-ethyl-N-isopropylamiloride. In the present study, the polymerase chain reaction (PCR) and library screening were used to clone a cDNA for one of the exchangers, based on homology with the recently isolated cDNA for a human growth factor-activatable Na(+)-H+ exchanger. There proved to be significant homology between the LLC-PK1 and human sequences, with nucleotide identities of 75, 93, and 85% in the 5'-untranslated, coding, and 3'-untranslated regions, respectively. The LLC-PK1 cDNA encodes a predicted protein of 818 amino acids with a relative molecular mass of 90,999, consisting of an amino-terminal hydrophobic region and a carboxy-terminal hydrophilic region; its deduced amino acid sequence shows 95% identity with that of the human protein. To investigate the localization of the encoded protein, antisera were generated against a synthetic oligopeptide from the hydrophobic region and a fusion protein from the carboxy-terminal hydrophilic domain. Indirect immunofluorescence and confocal microscopy revealed that the antisera labeled the basolateral but not the apical membrane of confluent LLC-PK1 cells. Labeling by the antipeptide antibody was specifically blocked by preincubation with the synthetic peptide and coincided exactly with the pattern produced by a monoclonal antibody against Na(+)-K(+)-ATPase. Thus, the LLC-PK1 cDNA encodes the basolateral Na(+)-H+ exchanger, which must differ structurally from the apical form, at least in the region of the oligopeptide and the fusion protein.
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PMID:cDNA cloning and immunolocalization of a Na(+)-H+ exchanger in LLC-PK1 renal epithelial cells. 166 Oct 81

We have analyzed the development of Na(+)-dependent hexose transport during differentiation and during polarization of LLC-PK1, an established cell line with characteristics of the proximal tubule. When cell-cell contact was disturbed by a low extracellular Ca2+ concentration or by a phorbol myristate acetate (PMA) treatment, the development of Na(+)-dependent hexose transport was completely inhibited. The effect of PMA on the development of hexose transport could be uncoupled from its effect on the tight junctions. The PMA concentration needed for the latter effect was approx. 10-fold higher than for the former. As the primary cause of the PMA effect, an influence on the cytoskeleton is suggested. In contrast to PMA, the concentration dependence of both phenomena on the extracellular Ca2+ concentration was almost the same. Moreover, the incorporation of hexose carriers in the plasma membrane could be induced by changing the extracellular CA2+ concentration from low to normal. We conclude that there is a relation between the formation of tight junctions and the development of the Na(+)-dependent hexose carrier, possibly because Ca(2+)-dependent cell adhesion molecules play a role in both phenomena. However, a direct relation between Ca(2+)-dependent elements of the tight junctions and the insertion of the hexose carrier can not be excluded. The Ca(2+)-dependent development seems to be a common characteristic of apical membrane proteins in contrast to the development of the basolateral membrane protein, (Na(+)+K+)-ATPase.
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PMID:Influence of PMA and a low extracellular Ca2+ concentration on the development of the Na(+)-dependent hexose carrier in LLC-PK1 cells. 167 54

Research on palytoxin focuses on its action as a tumor promoter and its ionophoretic action in cell membranes. The first property is unusual because palytoxin is not a protein kinase C activator. The second property is remarkable in that it may require interaction with the Na(+)-K(+)-adenosinetriphosphatase (ATPase). Our studies here with palytoxin exposure to the LLC-PK1 renal epithelial cells have yielded the following results: 1) unlike protein kinase C-activating tumor promoters (tetradecanoylphorbol 12,13-acetate or teleocidin), palytoxin does not produce a specific effect on the tight junctions between epithelia; 2) palytoxin instead produces an irreversible cytotoxic effect characterized by a pronounced cell swelling associated with sharply elevated levels of intracellular Na+ and decreased levels of intracellular K+; 3) these fluctuations in intracellular Na+ and K+ levels are explained by marked elevations in the membrane flux of 22Na+ and 86Rb+; 4) the electrophysiological reflection of these altered ion fluxes is a pronounced depolarization of the cell sheet if palytoxin is presented to the basolateral cell surface and a pronounced hyperpolarization (due to sharply elevated apical Na+ flux and transepithelial short-circuit current) if palytoxin is administered apically; 5) the apical effect of palytoxin can be blocked by apical ouabain; and 6) this apical effect of palytoxin decreases as a function of the age of the cell sheet. This first report of palytoxin action in a polar epithelial cell system provides additional evidence for palytoxin effects being mediated by contact with the Na(+)-K(+)-ATPase. It also adds to a growing literature suggesting the existence of Na(+)-K(+)-ATPase in the apical cell surface of epithelia under certain conditions.
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PMID:Effects of apical vs. basolateral palytoxin on LLC-PK1 renal epithelia. 167 41

Enhanced Na+ and water reabsorption by proximal tubular epithelial cells plays an important role in the development of systemic hypertension associated with cyclosporine immunosuppression. Since such Na+ reabsorption is subserved by sodium-potassium adenosine triphosphatase (Na-K ATPase), the current study compared the acute effects of hydrocortisone (H), cyclosporine, and FK506 on cultured LLC-PK1 cell viability and on Na-K ATPase activity. Phospholipase-C (PL-C) activity was also investigated because of its possible regulatory effect on Na-K ATPase activity. Culture medium containing low (5 nM, 4.1 ng/ml) or high (10 nM) concentrations of FK506 plus cyclosporine at 415 microM (500 ng/ml) resulted in cell death, whereas cyclosporine concentrations of 83 microM plus 5 nM or 10 nM FK506, or isolated use of the two drugs at high dosages, did not affect cell viability. As compared with controls, cyclosporine increased Na-K ATPase activity, particularly with addition of H (P less than 0.01). In contrast, FK506 reduced the specific activity of both PL-cyclosporine and Na-K ATPase (P less than 0.001-0.01); addition of H to FK506 resulted in an even greater fall in both the enzyme activities (P less than 0.001). Na-K ATPase activity increased in cell homogenates briefly incubated with cyclosporine in the ATPase reaction mixture (P less than 0.05) while FK506 reduced such enzyme activity (P less than 0.05), suggesting a direct effect of these agents on pump activity. These data in LLC-PK1 cells pocessing proximal tubular epithelial cell characteristics indicate that the combined use of cyclosporine plus FK506 may be very deleterious to viability in such cells. The opposing effects of cyclosporine and FK506 on PL-cyclosporine and Na-K ATPase activities and the possible potentiating effect of H on such responses are speculated to affect Na+ and water homeostasis in a manner that may explain differences in systemic blood pressure due to these agents.
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PMID:Phospholipase-C and Na-K ATPase activation by cyclosporine and FK506 in LLC-PK1, cells. Possible implications in blood pressure regulation. 171 43

Vacuolar proton pumps acidify several intracellular membrane compartments in the endocytic pathway. We have examined the distribution of the vacuolar H+ ATPase in LLC-PK1 cells and the structure of the biosynthetically labeled enzyme in membrane fractions enriched for endosomes or lysosomes. LLC-PK1 cells were allowed to internalize cytochrome c-coated colloidal gold as a marker for endocytic compartments. Proton pumps were identified in these cells by staining the cells with a monoclonal antibody against the vacuolar pump detected with either immunogold or immunoperoxidase techniques. H+ ATPase labeling was seen on structures resembling endosomes and lysosomes, but not on Golgi or plasma membrane. To examine the structure of the H+ ATPase in these compartments, we labeled LLC-PK1 cells for 24 h with [35S]methionine and used a Percoll gradient to obtain fractions enriched for endosomes or lysosomes. H+ ATPase immunoprecipitated from both fractions with monoclonal anti-H+ ATPase antibodies had labeled polypeptides of 70, 56, and 31 kDa. On two-dimensional gels, a comparison of the H+ ATPase from the endosomal and lysosomal fractions revealed that the 70-, 56-, and 31-kDa subunits were similar in both fractions. The results show that the vacuolar H+ ATPase in these cells is distributed primarily in endosomes and lysosomes and that the structure of the enzyme is similar in both compartments.
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PMID:Distribution and structure of the vacuolar H+ ATPase in endosomes and lysosomes from LLC-PK1 cells. 182 36


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